The objective of the research described in this proposal is to understand how an intracellular parasite gains entry into its host, evades the defenses of the host cell and multiplies in the host cell. Legionella pneumophila will be used as a model intracellular parasite. This organism enters human phagocyte cells (monocytes and macrophages), evades their microbicidal defenses, grows exponentially, and eventually kills the phagocytic cell. The advantage of using L. pneumophila as a model is that this organism is a Gram-negative bacterium that can be cultivated easily on bacterial growth media. Genetic techniques to transfer DNA from one Legionella strain to another as well as to E. coli will be developed. A rudimentary genetic map of Legionella will be constructed. A variety of Legionella mutants including those that are unable to kill human monocytes will be isolated. It is anticipated that these mutants will fall into three broadly defined categories: (i) those unable to enter by "coiling" phagocytosis, (ii) those which enter but are killed by the monocytes, and (iii) those which enter and survive but are unable to grow within the monocyte. The genetic defects in these mutants will be studied. Complementation analysis and mapping experiments will be used to define the genes that are responsible for entry of Legionella into phagoctyes, the ability of Legionella to evade the microbicidal factors present in monocytes, and the capacity to grow within monocytes.